IRBIT, a Novel Inositol 1,4,5-Trisphosphate (IP3) Receptor-bindingProtein, Is Released from the IP3 Receptor uponIP3 Binding to the Receptor*

Received for publication, October 3, 2002, and in revised form, January 9, 2003Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M210119200

Hideaki Ando‡§¶, Akihiro Mizutani‡�, Toru Matsu-ura‡, and Katsuhiko Mikoshiba‡§�

From the ‡Division of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai,Minato-ku, Tokyo 108-8639, Japan, the §Laboratory for Developmental Neurobiology, Brain Science Institute,Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan,and the �Calcium Oscillation Project, International Cooperative Research Project (ICORP), Japan Scienceand Technology Corporation, 3-14-4 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated Ca2� channels on intracellular Ca2�

stores. Herein, we report a novel protein, termed IRBIT(IP3R binding protein released with inositol 1,4,5-trisphosphate), which interacts with type 1 IP3R (IP3R1)and was released upon IP3 binding to IP3R1. IRBIT waspurified from a high salt extract of crude rat brain mi-crosomes with IP3 elution using an affinity column withthe huge immobilized N-terminal cytoplasmic region ofIP3R1 (residues 1–2217). IRBIT, consisting of 530 aminoacids, has a domain homologous to S-adenosylhomocys-teine hydrolase in the C-terminal and in the N-terminal,a 104 amino acid appendage containing multiple poten-tial phosphorylation sites. In vitro binding experimentsshowed the N-terminal region of IRBIT to be essentialfor interaction, and the IRBIT binding region of IP3R1was mapped to the IP3 binding core. IP3 dissociatedIRBIT from IP3R1 with an EC50 of �0.5 �M, i.e. it was 50times more potent than other inositol polyphosphates.Moreover, alkaline phosphatase treatment abolishedthe interaction, suggesting that the interaction was du-alistically regulated by IP3 and phosphorylation. Immu-nohistochemical studies and co-immunoprecipitationassays showed the relevance of the interaction in aphysiological context. These results suggest that IRBITis released from activated IP3R, raising the possibilitythat IRBIT acts as a signaling molecule downstreamfrom IP3R.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate inresponse to cell surface receptor activation leads to the produc-tion of an intracellular second messenger, inositol 1,4,5-trisphosphate (IP3).1 IP3 mediates the release of Ca2� from

intracellular Ca2� storage organelles, mainly the endoplasmicreticulum, by binding to its receptor (IP3R). In these IP3/Ca2�

signaling cascades, IP3R works as a signal converter from IP3

to Ca2� (1–3).IP3R is a tetrameric intracellular IP3-gated Ca2� release

channel (3, 4). There are three distinct types of IP3R in mam-mals (5–7). Type 1 IP3R (IP3R1) is highly expressed in thecentral nervous system, particularly in the cerebellum (8, 9).Mouse IP3R1 is composed of 2749 amino acids (5), and isdivided into three functionally distinct regions: the IP3-bindingdomain near the N terminus, the channel-forming domain withsix membrane-spanning regions close to the C terminus, andthe regulatory domain separating the two regions (10, 11).Deletion mutagenesis analysis of the IP3-binding domain hasshown that residues 226–578 of IP3R1 are close to the mini-mum for specific and high affinity ligand binding, thus as-signed to the IP3 binding core (12). The precise gating mecha-nism of IP3R triggered by IP3 remains unclear, but IP3 bindinginduces a substantial but as yet undefined conformationalchange, which may cause channel opening (10). Besides thischannel opening, such IP3-induced conformational change hasbeen assumed to be responsible for degradation of IP3R (13, 14).

The increase in the cytoplasmic Ca2� concentration resultingfrom IP3R activation regulates the activities of thousands ofdownstream targets that play key roles in many aspects ofcellular processes, including fertilization, development, prolif-eration, secretion, and synaptic plasticity (1, 2, 15). To controlsuch a vast array of cell functions, Ca2� signals need to beprecisely regulated in terms of space, time and amplitude (2,15). Such a complex regulation of Ca2� signals has been partlyattributed to the diversity of IP3R isoform expression, assemblyof heterotetrameric complexes of IP3R isoforms, subcellulardistributions of IP3R, and regulation of IP3R by Ca2� itself,ATP, and phosphorylation (3, 4, 16). IP3R channels are alsoregulated by their interacting proteins (4, 17), including cal-modulin (18, 19), FKBP12 (Refs. 20–22, but also see Refs. 23and 24), calcineurin (Refs. 21 and 25, but also see Refs. 23 and24), ankyrin (26–28), sigma-1 receptor (28), chromogranins Aand B (29–31), IRAG (32), Fyn (33), and BANK (34). Moreover,a family termed CaBP has been shown to interact with IP3R ina Ca2�-dependent manner, and to directly activate IP3R in theabsence of IP3 (35). IP3R has also been demonstrated to bephysically coupled to its upstream or downstream signalingmolecules by protein-protein interactions. For example, IP3R is

* This work was supported by grants from the Ministry of Education,Science, Sports and Culture of Japan and from RIKEN, the BrainScience Institute of Saitama, Japan. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AB092504.

¶ To whom correspondence should be addressed. Tel.: 81-3-5449-5319; Fax: 81-3-5449-5420; E-mail: hando@ims.u-tokyo.ac.jp.

1 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R,inositol 1,4,5-trisphosphate receptor; IP3R1, type 1 inositol 1,4,5-trisphosphate receptor; mGluRs, metabotropic glutamate receptors;B2Rs, B2 bradykinin receptors; IRBIT, IP3R-binding protein releasedwith inositol 1,4,5-trisphosphate; GST, glutathione S-transferase; GFP,green fluorescent protein; IP2, inositol 4,5-bisphosphate; IP4, inositol

1,3,4,5-tetrakisphosphate; IP6, inositol 1,2,3,4,5,6-hexakisphosphate;PBS, phosphate-buffered saline; FRET, fluorescence resonance energytransfer; PIPES, 1,4-piperazinediethanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 12, Issue of March 21, pp. 10602–10612, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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coupled with group 1 metabotropic glutamate receptors(mGluRs) via the Homer family of proteins (36) and with B2

bradykinin receptors (B2Rs) by an unknown mechanism (37).Activations of mGluRs and B2Rs lead to the production of IP3 inproximity to IP3R, the result being efficient and specific signalpropagation. Another example is Trp3, a candidate for plasmamembrane Ca2� channels regulated by intracellular Ca2� storedepletion (capacitative calcium entry channels). IP3R has beenshown to interact with Trp3 directly, and to activate it via aconformational coupling mechanism (38, 39). These protein-protein interactions are supposed to regulate the IP3/Ca2� sig-naling pathway and contribute to the specificity of intracellularCa2� dynamics.

To gain further insights into regulation of the IP3/Ca2� sig-naling pathway, we searched for IP3R-binding proteins. Inparticular, we focused on molecules that interact with IP3R ina manner regulated by IP3, because such molecules may rec-ognize the conformational change in IP3R induced by IP3 bind-ing, and/or may function as novel upstream or downstreamsignaling molecules of IP3R. For this purpose, we used anaffinity column conjugated with the N-terminal 2217 aminoacid residues of IP3R1 containing most of the large cytoplasmicregion of the receptor molecule. By eluting bound proteins withIP3 from this affinity column, we identified a novel IP3R-bind-ing protein, IRBIT (IP3R binding protein released with inositol1,4,5-trisphosphate). IRBIT bound to IP3R1 in vitro and in vivo,and co-localized intensively with IP3R1. Moreover, IRBIT wasreleased from IP3R1 at a physiological concentration of IP3. Onthe basis of these results, we consider herein the role of IRBITin IP3/Ca2� signaling.

EXPERIMENTAL PROCEDURES

Preparation of IP3R1 Affinity Column—The cDNA encoding the N-terminal region of mouse IP3R1 (residues 1–225) was inserted intoglutathione S-transferase (GST) fusion vector pGEX-KG (40). The GST-IP3R1 (1–225) fragment was subcloned into the baculovirus transfervector pBlueBac4.5 (Invitrogen). The 3�-region downstream from theSmaI site of GST-IP3R1-(1–225) was replaced with the SmaI-EcoRIfragment of mouse IP3R1 (corresponding to residues 79–2217) to gen-erate GST-IP3R1-(1–2217) (termed GST-EL, for the EcoRI Large frag-ment) construct. GST alone was subcloned into pBlueBac4.5 as a con-trol. Sf9 cells were cultured in TNM-FH medium supplemented with10% fetal bovine serum at 27 °C. Recombinant baculoviruses carryingGST-EL or GST were generated with the Bac-N-BlueTM transfection kit(Invitrogen) according to the manufacturer’s protocols. GST-EL andGST were expressed in 2 � 108 Sf9 cells by infecting recombinantbaculoviruses at a multiplicity of infection of 5, and incubating for 48 h.Cells were harvested and stored at �80 °C. Frozen cells were sus-pended in 10 ml of 10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA,1 mM 2-mercaptoethanol, 0.1% Triton X-100, and protease inhibitors (1mM phenylmethylsulfonyl fluoride, 10 �M leupeptin, 2 �M pepstatin A,and 10 �M E-64), and were homogenized with a glass-Teflon homoge-nizer (1000 rpm, 10 strokes). The homogenate was centrifuged at20,000 � g for 30 min. The supernatant was incubated with 3 ml ofglutathione-Sepharose 4B (Amersham Biosciences) for 3 h at 4 °C. Afterwashing eight times with 40 ml of 10 mM Hepes (pH 7.4), 250 mM NaCl,2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.1% Triton X-100, GST-ELor GST coupled with glutathione-Sepharose was packed into columnsand equilibrated with 10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM

EDTA, 1 mM 2-mercaptoethanol, and 0.1% Triton X-100. About 5 mg ofGST-EL was immobilized.

Purification and Partial Amino Acid Sequencing of IRBIT—Adult ratcerebella (�5 g) were homogenized in 45 ml of homogenizer buffer (10mM Hepes (pH 7.4), 320 mM sucrose, 2 mM EDTA, 1 mM 2-mercapto-ethanol, and protease inhibitors) with a glass-Teflon homogenizer (950rpm, 10 strokes), and the homogenate was centrifuged at 1,000 � g for10 min. The supernatant (S1 fraction) was centrifuged at 100,000 � gfor 60 min to obtain the cytosolic fraction (the supernatant) and thecrude microsome (the pellet). The crude microsome was homogenized in25 ml of homogenizer buffer containing 500 mM NaCl with a glass-Teflon homogenizer (1,200 rpm, 10 strokes), incubated on ice for 15 min,and centrifuged at 100,000 � g for 60 min to obtain the high salt extract(the supernatant) and the stripped-crude microsome (the pellet). The

high salt extract was diluted five times with 10 mM Hepes (pH 7.4), 2mM EDTA, 1 mM 2-mercaptoethanol, 0.01% Brij 35, and protease inhib-itors. The diluted high salt extract was precleared with glutathione-Sepharose and loaded onto a GST-EL affinity column equilibrated withbinding buffer (10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA, and1 mM 2-mercaptoethanol). The GST column was used as a control. Thecolumns were washed with 20 column volumes of binding buffer, andbound proteins were eluted with binding buffer containing 50 �M IP3

(Dojindo) and 0.05% Brij 35. The eluted material was concentrated,separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 10%gel, and stained with Coomassie Brilliant Blue. The 60-kDa proteinband was excised from the gel and digested with lysyl endopeptidase(Wako) essentially according to the previously described method (41).The polypeptides were separated by a C18 reversed-phase column(�RPC C2/C18 SC 2.1/10, Amersham Biosciences) connected on aSMART system (Amersham Biosciences). The amino acid sequence ofeach peptide was determined by 494 procise protein sequencer (AppliedBiosystems). Two peptide sequences, N-YSFMATVTK-C and N-QIQ-FADDMQEFTK-C were obtained.

cDNA Cloning of IRBIT—BLAST searches of two peptide sequencesderived from the 60-kDa protein against the non-redundant data baserevealed that these sequences match the sequence of a human cDNAdeposited in a patent (GenBankTM accession number CAC09285).Based on the data bases of mouse expressed sequence tags (accessionnumber AW229870 and BE282170) homologous to this cDNA, primers(5�-ATGTCGATGCCTGACGCGATGC-3� and 5�-GCGTGGTTCATGTG-GACTGGTC-3�) were synthesized. cDNA of IRBIT was amplified bypolymerase chain reaction (PCR) using mouse cerebellum oligo(dT)-primed, first-strand cDNA as a template. PCR product was cloned intopBluescript II KS(�) (Stratagene) and sequenced. Sequences of threeindependent clones were confirmed.

Preparation of Recombinant Proteins—The cDNA encoding the N-terminal region (residues 1–104) of IRBIT was subcloned into the bac-terial hexahistidine (His6) fusion vector pET-23a(�) (Novagen) to gen-erate the IRBIT-(1–104)-His6 construct. The same cDNA was subclonedinto the GST fusion vector pGEX-4T-1 (Amersham Biosciences) to gen-erate the GST-IRBIT-(1–104) construct. The cDNA fragments corre-sponding to the amino acid residues 1–225, 1–343, 341–923, 600–1248,916–1581, and 1553–1943 of mouse IP3R1 were inserted into pGEX-KGto generate the GST-Ia, GST-Iab, GST-IIab, GST-IIbIIIa, GST-IIIab,and GST-IV construct, respectively. Residues 1593–2217 of mouseIP3R1 were inserted into pGEX-4T-1 to generate the GST-IV-Va con-struct. These fusion proteins were expressed in Escherichia coli.GST-EL was expressed in Sf9 cells as described above. ExpressedIRBIT-(1–104)-His6 was purified using ProBond resin (Invitrogen).GST fusion proteins were purified using glutathione-Sepharose. GST-IbIIa (residues 224–604 of mouse IP3R1) and its site-directed mutantsK508A and R441Q were described previously (Ref. 42, GST-IbIIa wastermed G224 therein).

Production of Affinity-purified Anti-IRBIT Antibody—A Japanesewhite rabbit was immunized with purified IRBIT-(1–104)-His6 by sub-cutaneous injection with the complete Freund’s adjuvant at 14-dayintervals. The anti-IRBIT antisera was affinity-purified by passingserum from the immunized rabbit over a GST-IRBIT-(1–104) columncovalently coupled with cyanogen bromide-activated Sepharose 4B(Amersham Biosciences), and specific antibodies bound to the columnwere eluted with 100 mM glycine-HCl (pH 2.5).

Subcellular Fractionation and Immunoblotting—Cerebrum, cerebel-lum, heart, lung, liver, kidney, thymus, spleen, testis, and ovary weredissected from the adult mouse and S1 fraction were obtained essen-tially as described above. The cytosol, the crude microsome, the highsalt extract, and the stripped-crude microsome of mouse cerebellumwere obtained essentially as described above. Proteins with the amountindicated were subjected to 10% SDS-PAGE and transferred onto poly-vinylidene difluoride membrane by electroblotting. After blocking,membranes were immunoblotted with anti-IRBIT antibody (1 �g/ml)for 1 h at room temperature, followed by horseradish peroxidase-con-jugated donkey anti-rabbit IgG (Amersham Biosciences). Immunoreac-tive bands were visualized with the enhanced chemiluminescence de-tection system (Amersham Biosciences).

Generation and Transfection of Expression Constructs—The cDNAencoding full-length IRBIT was subcloned into the pcDNA3 (Invitro-gen). The cDNA encoding full-length IRBIT or its deletion mutants(residues 1–277, 1–104, and 105–530) were subcloned into thepEGFP-C1 (Clontech) to generate green fluorescent protein (GFP) fu-sion protein constructs. Mouse IP3R1 expression vector pBact-ST-neoB-C1 was described previously (43). COS-7 cells were cultured inDulbecco’s modified Eagle’s medium with 10% fetal bovine serum, pen-

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icillin, and streptomycin at 37 °C. Transient transfections were per-formed using TransIT transfection reagents (Mirus) according to themanufacturer’s instruction. Transfected cells were processed for immu-noblotting, pull-down experiments, or immunostaining 2 days aftertransfection.

In Vitro Binding Experiments—Mouse cerebellar cytosolic fractionwas diluted two times with 10 mM Hepes (pH 7.4), 200 mM NaCl, 2 mM

EDTA, 1 mM 2-mercaptoethanol, and 0.02% Triton X-100. The high saltextract was diluted five times with 10 mM Hepes (pH 7.4), 2 mM EDTA,1 mM 2-mercaptoethanol, and 0.01% Triton X-100. Diluted fractions(the final NaCl concentration of both fractions was 100 mM) wereincubated with 20 �g of GST-EL or GST for 2 h at 4 °C. After adding 10�l of glutathione-Sepharose and another 2-h incubation, the resins werewashed five times with wash buffer (10 mM Hepes, pH 7.4, 100 mM

NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.01% Triton X-100),and bound proteins were eluted with 20 mM glutathione. Eluted pro-teins were analyzed by Western blotting with anti-IRBIT antibody.

For dephosphorylation, the diluted high salt extract was incubatedwith or without bacterial alkaline phosphatase (Toyobo) in the presenceof 2 mM MgCl2 for 30 min at 37 °C after which 5 mM EDTA was added,and the sample was processed for pull-down assay as described above.

For the dissociation experiments, IRBIT in the diluted high saltextract was pulled down with GST-EL and washed as described above,and resins were added in 100 �l of wash buffer containing IP3, inositol4,5-bisphosphate (IP2) (Dojindo), inositol 1,3,4,5-tetrakisphosphate(IP4) (Calbiochem), inositol 1,2,3,4,5,6-hexakisphosphate (IP6) (Calbio-chem), or ATP (Amersham Biosciences) (0.1, 0.3, 1, 3, 10 �M, each).After incubation on ice for 10 min, samples were centrifuged at 10,000rpm for 1 min, and the supernatant was subjected to immunoblotanalysis with anti-IRBIT antibody or goat anti-GST antibody (Amer-sham Biosciences). For quantitation, Alexa 680-conjugated goat anti-rabbit IgG (Molecular Probes) was used as a secondary antibody. In-tensity of fluorescence of immunoreactive bands of IRBIT wasmeasured using Odyssey infrared imaging system (Aloka). Quantita-tive data (the mean � S.D. from at least three independent experi-ments) are expressed as percentage of the amount of IRBIT in the 10 �M

IP3 eluate.For the determination of the IRBIT binding region and the critical

amino acids of IP3R1, the diluted high salt extract were processed forpull-down assay with 100 pmol of GST, GST-EL, GST-Ia, GST-Iab,GST-IbIIa, GST-IIab, GST-IIbIIIa, GST-IIIab, GST-IV, GST-IV-Va,K508A, or R441Q as described above and analyzed by Western blottingwith anti-IRBIT antibody.

For the determination of the IP3R1-interacting region of IRBIT,COS-7 cells expressing GFP-tagged full-length IRBIT or its truncatedmutants were lysed in lysis buffer (10 mM Hepes pH 7.4, 100 mM NaCl,2 mM EDTA, 1 mM 2-mercaptoethanol, 0.5% Nonidet P-40, and proteaseinhibitors) for 30 min at 4 °C, followed by centrifugation (100,000 � g,30 min). The supernatants were processed for pull-down assay withGST-EL or GST as described above, and bound proteins were subjectedto immunoblot analysis with anti-GFP antibody (Medical & BiologicalLaboratories).

Indirect Immunofluorescence and Confocal Microscopy—TransfectedCOS-7 cells grown on glass coverslips were washed once in phosphate-buffered saline (PBS), fixed in 4% formaldehyde in PBS for 15 min,permeabilized in 0.1% Triton X-100 in PBS for 5 min, and blocked inPBS containing 2% normal goat serum for 60 min at room temperature.For washing out cytosolic proteins, transfected cells were washed oncein PBS, permeabilized in ice-cold permeabilization buffer (80 mM

PIPES, pH 7.2, 1 mM MgCl2, 1 mM EGTA, and 4% polyethylene glycol)containing 0.1% saponin for 10 min on ice, and washed twice withice-cold permeabilization buffer before fixation. Cells were then stainedwith rabbit anti-IRBIT antibody (1 �g/ml for 60 min at room tempera-ture) and rat anti-IP3R1 antibody 18A10 (44) overnight at 4 °C. Follow-ing four 5-min PBS washes, Alexa 488-conjugated goat anti-rabbit IgGand Alexa 594-conjugated goat anti-rat IgG (Molecular Probes) wereapplied for 45 min at 37 °C. Following four 5-min PBS washes, thecoverslips were mounted with Vectashield (Vector Laboratories) andobserved under IX-70 confocal fluorescence microscopy (Olympus) witha �60 objective.

Immunoprecipitation—Immunoprecipitation was performed as de-scribed (45) with modifications. Adult mouse cerebellum was homoge-nized in 10 volumes of 4 mM Hepes (pH 7.4), 320 mM sucrose, andprotease inhibitors with a glass-Teflon homogenizer. The homogenatewas centrifuged at 800 � g for 10 min, and the supernatant wassubjected to another centrifugation at 9000 � g for 15 min. The super-natant from the second centrifugation was solubilized in 1% sodiumdeoxycholate at 36 °C for 30 min, followed by adding 0.1 volume of 1%

Triton X-100 in 50 mM Tris-HCl (pH 9.0), and the preparation wascentrifuged at 100,000 � g for 10 min. The supernatant was incubatedwith 5 �l of protein G-Sepharose 4 fast flow (Amersham Biosciences) for2 h at 4 °C to clarify nonspecific binding to the protein G beads. At thesame time, 3 �g of rabbit anti-IRBIT antibody, control rabbit IgG, ratanti-IP3R1 antibody 10A6 (46), control rat IgG, mouse anti-IP3R2 an-tibody KM1083 (47), or control mouse IgG was preincubated with 5 �lof protein G beads for 2 h, and the protein G-antibody complex was spundown at 3,000 rpm for 2 min. The clarified supernatant was then addedto the antibody-bound protein G beads, and the mixture was incubatedfor 2 h at 4 °C. Beads were washed five times with 10 mM Hepes (pH7.4), 100 mM NaCl, and 0.5% Triton X-100 and analyzed by Westernblotting with anti-IRBIT antibody, mouse anti-IP3R1 antibody KM1112(47), KM1083, or mouse anti-IP3R3 antibody KM1082 (47).

RESULTS

Purification and cDNA Cloning of a Novel IP3R-interactingProtein—To identify IP3R-interacting molecules, we used aGST fusion protein of the N-terminal 2217 amino acids ofmouse IP3R1 (GST-EL). This region is the large cytoplasmicportion of IP3R1 containing the IP3 binding domain and regu-latory domain (10, 11). GST-EL or GST was expressed using abaculovirus/Sf9 cell system and conjugated to glutathione-Sepharose. The extract with a high salt buffer (containing 500mM NaCl) from crude rat cerebellar microsomes, which wasthought to be enriched with peripherally membrane-bound pro-teins, was loaded onto a glutathione-Sepharose affinity columnon which GST-EL or GST was immobilized. To detect proteinsthat were dissociated from IP3R in the presence of IP3, theproteins bound to the affinity columns were eluted by additionof 50 �M IP3. A protein with a mass of about 60 kDa wasdetected in the 50 �M IP3-eluate from the GST-EL column (Fig.1A), but not from the GST column (data not shown). Twopeptide sequences derived from the 60-kDa protein were deter-mined. BLAST searches of non-redundant databases revealedthat these two sequences matched the sequence of a humancDNA deposited in a patent. On the basis of sequence informa-tion on mouse expressed sequence tags homologous to thiscDNA, the cDNA of the 60-kDa protein was obtained by reversetranscriptase-PCR from the mouse cerebellum. The predictedamino acid sequence of the cloned cDNA revealed a proteincomposed of 530 amino acid residues (Fig. 1B), with a calcu-lated molecular mass of 58.9 kDa, which was close to its ap-parent molecular mass of 60 kDa estimated by SDS-PAGE (Fig.1A). We designated the 60-kDa protein IRBIT.

Homology analysis of the deduced amino acid sequence ofIRBIT revealed the C-terminal region (residues 105–530) to behomologous (51% identical, 74% similar) to the methylationpathway enzyme S-adenosylhomocysteine hydrolase (EC3.3.1.1.) (48) (Fig. 1, C and D). An appendage of the N-terminalregion (residues 1–104) of IRBIT had no homology with re-ported proteins and contained a serine-rich region (residues62–103) (Fig. 1, B and D). Motif searches of the IRBIT sequencerevealed the presence of a putative coiled-coil motif (residues111–138) and a putative NAD� binding region (residues 314–344) (Fig. 1, B and D). There were 17 potential phosphorylationsites for protein kinases such as casein kinase II, PKC, PKA/PKG, and tyrosine kinases, out of which seven sites were con-centrated in the N-terminal region (Fig. 1B). Neither putativemembrane-spanning regions nor signal sequences were found.Recently, a mRNA expressed in dendritic cells was cloned froma human cDNA library, and it was named DCAL (49), but itsphysiological function was not addressed. The 100% identicalamino acid sequences of IRBIT and DCAL indicate that IRBITis a mouse homologue of human DCAL.

Although IRBIT was homologous with S-adenosylhomocys-teine hydrolase, which catalyzes the reversible hydrolysis ofS-adenosylhomocysteine to adenosine and homocysteine, re-combinant IRBIT expressed in bacteria had no enzyme activi-

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ties in the hydrolysis direction, nor had any effects on theenzyme activity of S-adenosylhomocysteine hydrolase (data notshown).

Tissue Distribution and Subcellular Localization of IR-BIT—We generated an affinity-purified antibody against theN-terminal region of the IRBIT (Fig. 1B, boxed). To confirm thespecificity of this antibody, we transfected the cDNA of IRBITinto COS-7 cells, and the whole cells lysates obtained wereanalyzed by immunoblotting with the anti-IRBIT antibody. Asshown in Fig. 2A, the anti-IRBIT antibody recognized only asingle protein with a size of �60 kDa. The molecular mass ofthe exogenously expressed IRBIT (Fig. 2A, lane 1) was thesame as that of the endogenous protein in COS-7 (Fig. 2A, lane3), confirming that the cDNA clone encodes the full-length

IRBIT protein. We examined the expression of IRBIT in severalmouse tissues by immunoblot analysis with this anti-IRBITantibody. IRBIT was detected ubiquitously, with the highestexpressions in the cerebrum and cerebellum (Fig. 2B).

Next, we investigated the subcellular distribution of IRBITby fractionation of the mouse cerebellum. IRBIT was present inboth the cytosolic and the crude microsome fraction (Fig. 2C,lanes 2 and 3, respectively). The crude microsome fraction wasfurther separated into a peripherally membrane-bound frac-tion (the fraction from which IRBIT was originally purified)and a stripped-membrane fraction, with the aforementionedhigh salt buffer. As shown in Fig. 2C, IRBIT in the crudemicrosome fraction was partially extracted with the high saltbuffer (Fig. 2C, lane 4). In contrast, IP3R1, which is an integral

FIG. 1. Purification and cDNA cloning of IRBIT. A, SDS-PAGE of IRBIT (indicated by arrow) purified from the high salt extract of cruderat cerebellar microsomes with the GST-EL column. IRBIT was eluted from the column with 50 �M IP3. The eluted material was concentrated,separated by SDS-PAGE on 10% gel and stained with Coomassie Brilliant Blue. The bands above 200 kDa and at 100–200 kDa were mostly leakedGST-EL and its degradation products, respectively because these bands were recognized by various anti-IP3R1 antibodies. B, the deduced aminoacid sequences of IRBIT. Two digested peptides obtained from the purified IRBIT are bold-underlined. The serine-rich region is dashed-underlined.The putative coiled-coil region is double-underlined. The putative NAD� binding site is underlined. Putative phosphorylation sites for caseinkinase II (closed circles), PKC (open circles), PKA/PKG (a closed square), and tyrosine kinases (open squares) are indicated above the sequences.The N-terminal region is boxed. C, sequence alignment of the C-terminal region of IRBIT and rat S-adenosylhomocysteine hydrolase (AHCY) (48).Identical residues (*) and similar residues (:) are indicated. Residues involved in substrate binding and NAD� binding of S-adenosylhomocysteinehydrolase are indicated by closed circles and open circles, respectively. D, schematic representation of the structure of IRBIT. NTR and CTRindicate the N-terminal region and the C-terminal region, respectively. Serine-rich region (SER), coiled-coil region (CC) and NAD� binding site(NAD) are indicated.

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membrane protein of the endoplasmic reticulum, was not ex-tracted (Fig. 2C, lower panel). These results indicate IRBITto be both a cytosolic and a peripherally membrane-boundprotein.

IRBIT in the High Salt Extract Interacted with IP3R1 andthe N-terminal Region of IRBIT Was Essential for Interaction—IRBIT was present in both the cytosolic and the peripherallymembrane-bound fraction of the mouse cerebellum (Fig. 2C).We investigated whether IRBIT in these fractions interactedwith IP3R1 in vitro employing GST pull-down techniques. Thecytosol and high salt extracts from crude mouse cerebellarmicrosomes were incubated with GST-EL or GST, and bindingof IRBIT to the recombinant proteins was analyzed by immu-noblotting with anti-IRBIT antibody. As shown in Fig. 3A,IRBIT in the high salt extract interacted with GST-EL (Fig. 3A,lane 6), but not with GST (Fig. 3A, lane 5). In contrast, IRBITin the cytosolic fraction did not interact with GST-EL (Fig. 3A,lane 3). The same result was obtained when both fractions weredialyzed against the same buffer, indicating that the differencewas due neither to a difference in buffer composition nor toexcluded small molecules (data not shown). We speculated thatthe difference might be attributable to a post-translationalmodification of IRBIT such as phosphorylation. To test thispossibility, we treated the high salt extract with alkaline phos-phatase, a nonspecific phosphatase, followed by incubationwith GST-EL or GST. As shown in Fig. 3B, IRBIT in the highsalt extract no longer interacted with GST-EL after phospha-tase treatment (Fig. 3B, lane 6). This result raises the possi-bility that phosphorylation of IRBIT may be necessary forassociation with IP3R1, although the possibility that phospho-rylation of other proteins may regulate the interaction betweenIRBIT and IP3R1 cannot be excluded.

To determine the region of IRBIT responsible for the inter-action with IP3R1, GST pull-down experiments were carriedout using GFP-tagged deletion mutants of IRBIT (Fig. 4A). Asshown in Fig. 4B, both GFP-IRBIT and GFP-IRBIT-(1–277)bound to GST-EL efficiently (Fig. 4B, lanes 3 and 6, respective-ly). Although GFP-IRBIT-(1–104) interacted with GST-EL, theinteraction was much weaker than those of GFP-IRBIT and

FIG. 3. IRBIT in the high salt extract but not in the cytosolinteracted with IP3R1 in vitro. A, mouse cerebellar cytosolic fraction(lanes 1–3) and the high salt extract of crude microsomes (lanes 4–6)were incubated with GST-EL (lanes 3 and 6) or GST (lanes 2 and 5).Bound proteins were pulled down with glutathione-Sepharose, elutedwith glutathione, and analyzed by Western blotting using anti-IRBITantibody (upper panel). GST-EL and GST pulled down with glutathi-one-Sepharose were visualized by staining with Coomassie BrilliantBlue (lower panel). B, the high salt extract of crude mouse cerebellarmicrosomes was incubated without (lanes 1–3) or with (lanes 4–6)alkaline phosphatase prior to pull down with GST-EL (lanes 3 and 6) orGST (lanes 2 and 5). IRBIT binding was analyzed as in A.

FIG. 2. Tissue distribution and subcellular fractionation of IRBIT. A, Western blot analysis of exogenously expressed and endogenousIRBIT. COS-7 cells were transiently transfected with IRBIT (lane 1) or mock control (lanes 2 and 3), and the whole cell lysates were analyzed byWestern blotting with anti-IRBIT antibody. In lane 3, 10� amounts of the lysate were loaded as compared with those in lanes 1 and 2. B, tissuedistribution of IRBIT. S1 fractions (2 �g of total protein) of adult mouse tissues were analyzed by Western blotting with anti-IRBIT antibody. C,subcellular fractionation of the mouse cerebellum. S1 fraction (lane 1) of mouse cerebella was centrifuged at 100,000 � g to obtain the cytosolicfraction (lane 2) and the crude microsomes (lane 3). The crude microsomes were extracted with the high salt buffer containing 500 mM NaCl andcentrifuged at 100,000 � g to obtain the peripherally membrane-bound fraction (lane 4) and the stripped-crude microsomes (lane 5). Upper, eachfraction (1 �g of total protein) was analyzed by Western blotting with anti-IRBIT antibody. Lower, each fraction (0.2 �g of total protein) wasanalyzed by Western blotting with anti-IP3R1 antibody.

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GFP-IRBIT-(1–277) (Fig. 4B, compare lanes 7 and 9 with lanes1 and 3 and lanes 4 and 6). In contrast, GFP-IRBIT-(105–530),which lacked the N-terminal region, and GFP alone did notinteract with GST-EL (Fig. 4B, lanes 12 and 15, respectively).These results demonstrate the N-terminal region of IRBIT tobe essential for the interaction with IP3R1, and the following�170 amino acids containing a coiled-coil structure might beimportant for stabilizing the interaction.

IRBIT Co-localized with IP3R1 on the Endoplasmic Reticu-lum in Transfected COS-7 Cells—To test whether IRBIT inter-acts with IP3R1 in intact cells, IRBIT and IP3R1 were co-expressed in COS-7 cells, and their distribution was analyzedby confocal immunofluorescence microscopy. IRBIT was dif-fusely distributed in the cytoplasm, with no immunoreactivityin the nucleus (Fig. 5A). Because IRBIT was shown to bepresent in both the cytosolic and the crude microsome fractionby biochemical fractionation (Fig. 2C), we attempted to visual-ize only the membrane-bound population of IRBIT. For thispurpose, we permeabilized plasma membranes of transfectedCOS-7 cells with saponin and washed out cytosolic IRBIT priorto fixation. As shown in Fig. 5B, in cells treated with saponin,localization of IRBIT on the reticular structure was revealed(Fig. 5B, left panels). The immunoreactivity of IRBIT exten-sively overlapped with that of IP3R1 (Fig. 5B, middle panels,and merged image right panels). The staining pattern of IP3R1was not altered by permeabilization with saponin (data notshown). Since IRBIT expressed alone showed a coarse distri-bution instead (data not shown), these results indicate thatIRBIT co-expressed with IP3R1 localized on the endoplasmicreticulum via the interaction with IP3R1. IP3R1 was expressedin COS-7 cells to a trace level, whereas type 2 IP3R (IP3R2) andtype 3 IP3R (IP3R3) were predominantly expressed (50, 51).

These endogenous IP3Rs showed again a coarse, not a reticular,distribution in COS-7 cells both in a previous report and in ourhands (Ref. 52 and data not shown, respectively). Furthermore,a complex of IRBIT and endogenous IP3R2/IP3R3 were re-vealed by co-immunoprecipitation assay (data not shown).Taken together, these findings support our idea that IRBITinteracted not only with IP3R1 but also with IP3R2 and IP3R3(see below).

When we transfected IP3R1 and GFP-IRBIT instead of IR-BIT and observed the fluorescence of GFP, essentially the sameresults were obtained (Fig. 5, C and D). To confirm the speci-ficity of co-localization, we transfected GFP-IRBIT-(105–530),which did not interact with GST-EL because of the lack of theN-terminal region (Fig. 4), with IP3R1 into COS-7 cells. Incontrast to GFP-IRBIT, GFP-IRBIT-(105–530) was distributedin the nucleus as well as the cytosol (Fig. 5E). IRBIT does notharbor predicted nuclear localization signals, and the reasonGFP-IRBIT-(105–530) localized in the nucleus is unclear atpresent. When the cytosolic population was washed out bypermeabilization, GFP-IRBIT-(105–530) localized only in thenucleus and did not co-localize with IP3R1 (Fig. 5F). This ob-servation is consistent with biochemical results indicating theN-terminal region of IRBIT to be necessary for binding toIP3R1 (Fig. 4B).

IRBIT Interacted with IP3R in Vivo—To demonstrate an invivo association between IRBIT and IP3R in native tissues, weperformed co-immunoprecipitation experiments using mousecerebellum. Cerebellar lysates were immunoprecipitated withanti-IRBIT antibody, and the immunoprecipitates were ana-lyzed by immunoblotting with anti-IP3R1, anti-IP3R2, or anti-IP3R3 antibody. All three IP3R isoforms were co-immunopre-cipitated by anti-IRBIT antibody, but not control IgG (Fig. 6A).In the reciprocal experiments, immunoprecipitation of IP3R1 orIP3R2 resulted in the co-precipitation of IRBIT (Fig. 6, B andC). IRBIT was not detected in the anti-IP3R3 precipitates,probably due to the inefficiency of immunoprecipitation withanti-IP3R3 antibody (data not shown). When we performedimmunoprecipitation assay using lysates of COS-7 cells trans-fected with IRBIT and IP3R3, in which most IP3R3 formshomotetramers (51), IRBIT was shown to interact with IP3R3(data not shown). As for IP3R2, essentially the same result wasobtained (data not shown). These results confirm IRBIT inter-acted with all IP3R isoforms in vivo.

Physiological Concentration of IP3 Selectively Dissociated IR-BIT from IP3R1—IRBIT was originally identified in theGST-EL column eluate with 50 �M IP3 (Fig. 1A), suggestingthat IP3 disrupted the interaction between IRBIT and IP3R1.However, 50 �M is a relatively high concentration comparedwith the physiological range of IP3, which was estimated to bea few micromolar after stimulation (53). Thus, we examinedthe dose-dependence of IP3 with which IRBIT was dissociatedfrom GST-EL, and its selectivity against other related inositolpolyphosphates. IRBIT in the high salt extract of crude mousecerebellar microsomes was pulled down with GST-EL, andeluted with 0.1–10 �M IP3, IP2, IP4, IP6, or ATP. As shown inFig. 7A, IP3 dissociated IRBIT from GST-EL most efficiently ina dose-dependent manner (Fig. 7Aa, lower panel). We con-firmed GST-EL to be undetectable in the IP3 eluates (Fig. 7Aa,upper panel), even with longer exposure (data not shown). TheEC50 (the concentration required for half-maximal dissociationof IRBIT from GST-EL) was �0.5 �M, which was within thephysiological IP3 concentration range (53) (Fig. 7B). IP3 disso-ciated IRBIT from GST-EL about 50 times more efficientlythan other inositol polyphosphates (Fig. 7, Ab–d and B). ATP,which has three phosphate groups like IP3, did not dissociateIRBIT from GST-EL even at 10 �M (Fig. 7, Ae and B). These

FIG. 4. The N-terminal region of IRBIT was essential for inter-action with IP3R1. A, schematic representation of the structure ofIRBIT and its GFP-tagged truncated mutants. B, GST pull-down assayfrom the lysates of COS-7 cells expressing GFP-IRBIT (lanes 1–3),GFP-IRBIT-(1–277) (lanes 4–6), GFP-IRBIT-(1–104) (lanes 7–9), GFP-IRBIT-(105–530) (lanes 10–12), and GFP (lanes 13–15). The lysates ofCOS-7 cells expressing each construct (input; I) were incubated withGST-EL (E) or GST (G). Bound proteins were pulled down with gluta-thione-Sepharose, eluted with glutathione, and subjected to immuno-blot analysis with anti-GFP antibody.

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results indicate that IRBIT was dissociated from IP3R1 selec-tively within the physiological concentration range of IP3.

IRBIT Interacted with the IP3 Binding Region of IP3R1 andLys-508 of IP3R1 Was Essential for Interactions with BothIRBIT and IP3—We investigated which region, the IP3 bindingregion or the regulatory region, of IP3R1 was necessary for theinteraction with IRBIT, using eight deletion mutants of IP3R1constructed as GST fusion proteins based on the domain struc-ture of IP3R1 (54) (Fig. 8A). As shown in Fig. 8B, GST-IbIIa

(residues 224–604), which contains the IP3 binding core region(residues 226–578) (12) bound to IRBIT to the same extent asGST-EL. In contrast, other GST fusion proteins, includingGST-Iab and GST-IIab, did not interact with IRBIT. Next, weperformed a site-directed mutagenesis analysis to determinethe IP3R1 amino acids important for the interaction with IR-BIT. Lys-508 of IP3R1 was a critical amino acid residue for IP3

binding (12), and substitution of Lys-508 of GST-IbIIa withalanine (K508A) resulted in an enormous loss of IP3 binding

FIG. 5. IRBIT co-localized with IP3R1 in transfected COS-7 cells. IP3R1 was transiently transfected into COS-7 cells with IRBIT (A andB), GFP-IRBIT (C and D), and GFP-IRBIT-(105–530) (E and F). The localization of the corresponding proteins was analyzed by indirectimmunofluorescence (IP3R1 and IRBIT) and fluorescence (GFP-IRBIT and GFP-IRBIT-(105–530)) confocal microscopy. B, D, and F, cells werepermeabilized in saponin and cytosolic proteins were washed out prior to fixation. Left panels show IRBIT (B), GFP-IRBIT (D), and GFP-IRBIT-(105–530) (F). Middle panels show IP3R1. Right panels show merged images of fluorescence from left and middle panels. B, lower panels are highermagnification images of upper panels. Scale bars, 10 �m.

FIG. 6. IRBIT associated with IP3Rin vivo. A, cerebellar lysates were immu-noprecipitated with anti-IRBIT or controlantibody. The immunoprecipitates weresubjected to SDS-PAGE followed by West-ern blotting with anti-IP3R1, anti-IP3R2,anti-IP3R3, or anti-IRBIT antibody. B,cerebellar lysates were immunoprecipi-tated with anti-IP3R1 or control antibody.The immunoprecipitates were subjectedto Western blotting with anti-IRBIT oranti-IP3R1 antibody. C, cerebellar lysateswere immunoprecipitated with anti-IP3R2 or control antibody. The immuno-precipitates were subjected to Westernblotting with anti-IRBIT or anti-IP3R2antibody. Arrowheads indicate immuno-globulin heavy chains.

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affinity (42). Conversely, R441Q, in which Arg-441 of GST-IbIIa was substituted for Gln, had higher IP3 affinity thanGST-IbIIa (42). GST pull-down assays using these recombinantproteins showed that IRBIT bound to GST-IbIIa and R441Q tothe same extent, but not to K508A (Fig. 8C). Taken together,these results indicate that IRBIT binds to the IP3-bindingregion of IP3R1 and that Lys-508 of IP3R1 is required for theinteraction with IRBIT as well as IP3, supporting the observa-tion that IP3 disrupts the interaction between IRBIT andIP3R1.

DISCUSSION

We screened IP3R1-binding proteins released from IP3R1 inthe presence of IP3 and identified a novel protein, IRBIT, froma high salt extract of crude cerebellar microsomes. IRBIT in-teracted with IP3R1 in vitro and in vivo, and co-localized ex-tensively with IP3R1 in the endoplasmic reticulum in trans-fected cells. These results strongly suggest that IRBITassociates with IP3R1 in basal states. Moreover, the physiolog-ical concentration of IP3, but not of other inositol polyphos-phates, dissociated IRBIT from IP3R1. IRBIT bound to the IP3

binding region of IP3R1, and Lys-508 of IP3R1 was essential for

the interactions with both IP3 and IRBIT. These results sug-gest that IRBIT is released from IP3R1 with IP3 produced inresponse to extracellular stimuli. Although many IP3R-bindingproteins have been reported (18–39), IRBIT is the first mole-cule for which the interaction with IP3R was shown to beregulated by IP3.

IRBIT is composed of two regions, the N-terminal region(residues 1–104) essential for the interaction with IP3R1, andthe C-terminal region (residues 105–530) homologous to S-adenosylhomocysteine hydrolase (48). Crystallographic studies(55, 56) and site-directed mutagenesis studies (57–60) havedetermined amino acid residues of S-adenosylhomocysteine hy-drolase involved in substrate binding or NAD� binding (Fig.1C). Although most of these residues were well conserved inIRBIT, we did not detect enzyme activity of recombinant IRBITexpressed in bacteria. We concluded that the IRBIT does nothave S-adenosylhomocysteine hydrolase activity, probably dueto substitution of amino acids important for substrate binding,such as Leu-54, Phe-302, and His-353 of S-adenosylhomocys-teine hydrolase (Fig. 1C), as discussed by another group (49).Domains that are homologous to certain enzymes, but arecatalytically inactive, such as the esterase domain of the neu-

FIG. 7. Physiological concentration of IP3 selectively dissoci-ated IRBIT from IP3R1. A, the high salt extract of crude mousecerebellar microsomes was incubated with GST-EL. Bound proteinswere pulled down with glutathione-Sepharose and eluted with gluta-thione (Glu) (a) or 0.1–10 �M IP3 (a), IP2 (b), IP4 (c), IP6 (d), or ATP (e).IRBIT in the eluates were analyzed with anti-IRBIT antibody andAlexa 680-conjugated secondary antibody (a, lower, and b–e). GST-ELin the glutathione and 0.1–10 �M IP3 eluate was analyzed with anti-GST antibody (a, upper). B, the intensity of the immunoreactive bandsof IRBIT was quantified by infrared imaging system, and relativeintensity was plotted against concentration. Results are shown as themean � S.D. from at least three independent experiments.

FIG. 8. IRBIT interacted with the IP3-binding region of IP3R1and Lys-508 was critical for this interaction. A, schematic repre-sentation of the structure of mouse IP3R1 and the recombinant GSTfusion proteins used in this study. The IP3 binding core region isindicated with a gray box. Putative membrane-spanning regions areindicated by solid vertical bars. Roman numbers below IP3R1 indicatethe domain structure determined by the limited trypsin digestion (54).Numbers above the lines represent corresponding amino acid numbers.B, determination of the IRBIT binding region of IP3R1. The high saltextract of crude mouse cerebellar microsomes was incubated with GSTfusion proteins described in A. Bound proteins were pulled down withglutathione-Sepharose, eluted with glutathione, and analyzed by West-ern blotting using anti-IRBIT antibody. C, site-directed mutagenesisanalysis. The high salt extract was processed for pull-down assay withGST-IbIIa, R441Q, and K508A as described in B. Bound proteins wereanalyzed by Western blotting using anti-IRBIT antibody (upper panel).GST fusion proteins pulled down with glutathione-Sepharose were vi-sualized by staining with Coomassie Brilliant Blue (lower panel).

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roligin family (61) and the carbonic anhydrase domain of re-ceptor tyrosine phosphatase � (62), are reportedly involved inprotein-protein interactions. The C-terminal region of IRBITmay be one such domain. However, the possibility that IRBIThas enzyme activity with a different substrate specificity can-not be excluded.

In vitro binding experiments and immunostaining studiesshowed the N-terminal region of IRBIT to be essential, thoughnot sufficient, for the interaction with IP3R1. The IRBIT-bind-ing region of IP3R1 was shown to be its IP3 binding region, andLys-508 of IP3R1, the critical amino acid for IP3 binding, wasrequired for this interaction. Based on mutagenesis analysis,Yoshikawa et al. (12) proposed that basic amino acid residues,including Lys-508, contribute to form a positively chargedpocket for binding to the three negatively charged phosphategroups of IP3. This model leads us to speculate that acidic orphosphorylated amino acid residues in the N-terminal region ofIRBIT may be involved in interaction with the positivelycharged IP3-binding pocket of IP3R1. This hypothesis is sup-ported by the following findings: 1) although IRBIT is a neutralprotein (calculated pI of 6.48), its N-terminal region is rela-tively acidic (calculated pI of 4.98), 2) seven potential phospho-rylation sites are concentrated in the N-terminal region ofIRBIT, and phosphorylation was supposed to be required forthe interaction, 3) Lys-508 of IP3R1 was essential for the in-teraction with IRBIT, 4) IP3 disrupted the interaction, and 5) ahigh salt buffer disrupted the interaction between IRBIT andGST-EL2 and extracted IRBIT from crude microsomes, indicat-ing that the interaction is dependent on an electrostatic bond.Deletion mutagenesis results also indicate that residues 105–277 of IRBIT, which contain a coiled-coil region, contribute tothe interaction. The crystal structure of the IP3 binding regionof mouse IP3R1 in the complex with IP3 was recently resolved(63). IP3 bound to the positively charged cleft of the IP3 bindingregion, and the side chain of Lys-508 formed the hydrogen bondwith the phosphate group at the 5-position of IP3 (63). Remark-ably, the C-terminal region of the IP3 binding domain contain-ing Lys-508 (residues 437–604) formed an armadillo repeat-like fold (63), which generally acts as a protein-proteininteraction motif (64). IRBIT may interact with IP3R1 via thismotif. However, the armadillo repeat-like fold is not sufficientfor interaction, since GST-IIab (residues 341–923 of IP3R1) didnot interact with IRBIT.

IRBIT was dissociated from IP3R1 selectively with IP3 at anEC50 of �0.5 �M. This EC50 value is higher than the Kd ofpurified IP3R1 for IP3 (Kd � 83–100 nM) determined by con-ventional IP3 binding assays (46, 65). This difference may beattributable to different buffer conditions because the IP3 bind-ing affinity of IP3R depends strongly on pH and ionic strength(66–68). Conventional IP3 binding assays were performed un-der optimal binding conditions, with a higher pH (8.0–8.3) anda lower ionic strength (salt-free). Surface plasmon resonancebiosensor studies using the N-terminal region of IP3R1 (resi-dues 1–604) demonstrated the Kd value determined under nearphysiological conditions (pH 7.4 and 150 mM NaCl) to be 336 nM

(68), i.e. �7.5-fold lower than the affinity determined by theconventional IP3 binding assay (69), and close to the EC50 (�0.5�M) required for the dissociation of IRBIT from GST-EL deter-mined at pH 7.4 and 100 mM NaCl. Therefore, taken togetherwith the findings that IRBIT bound to the IP3 binding region ofIP3R1 and that both IRBIT and IP3 were dependent on Lys-508of IP3R1 for the interaction, these results indicate that IRBITis released from IP3R1 upon IP3 binding to IP3R1, probably viaa competitive mechanism.

Phosphorylation, as well as IP3, is considered to regulate theinteraction between IRBIT and IP3R. In vitro binding experi-ments showed IRBIT extracted from the membrane fractionwith a high salt buffer to interact with IP3R1, whereas IRBITin the cytosolic fraction did not. The difference in the phospho-rylation state of IRBIT may account for this discrepancy, be-cause alkaline phosphatase treatment of the high salt extractdisrupted the interaction between IRBIT and IP3R1. IRBIT has17 potential phosphorylation sites, and seven of these sites areconcentrated in the N-terminal region, which is necessary forthe interaction with IP3R1. These findings raise the possibilitythat the dephosphorylated form of IRBIT is free in the cytosol,whereas the phosphorylated form is membrane-bound via theinteraction with IP3R1, although we could not rule out thepossibility that phosphorylation of other proteins may regulatethe interaction. We propose that the interaction between IRBITand IP3R1 is dualistically regulated by IP3 and, either directlyor indirectly, by phosphorylation. Further studies are needed todetermine whether or not the interaction is regulated by directphosphorylation of IRBIT.

Using the detector cell/capillary electrophoresis system,Luzzi et al. (53) estimated intracellular IP3 concentrations be-fore and after stimulation to be tens of nanomolar and a fewmicromolar, respectively. Because the EC50 of IP3 (�0.5 �M)required for the dissociation of IRBIT from IP3R1 was betweenthese concentrations, IRBIT was assumed to be released fromIP3R1 after IP3 production has been induced by extracellularstimuli. What is the physiological significance of the dissocia-tion of IRBIT from IP3R1 after stimulation and what is thefunction of IRBIT? We propose four possible roles of IRBIT.First, IRBIT may modulate the channel activity of IP3R1. Re-cently, Yang et al. (35) showed that CaBP family members canact as direct ligands of IP3R. Interestingly, the CaBP-bindingregion of IP3R was within its 600 N-terminal residues (35),which also contain the IRBIT-binding region. Considering ourpreliminary data showing that IRBIT does not directly modu-late the channel activity of IP3R,2 IRBIT may block the bindingof CaBP to IP3R1 and inhibit IP3-independent activation ofIP3R1. Second, IRBIT may regulate the stability of IP3R. IP3-generating stimuli cause degradation of IP3R (13, 14, 50, 70–72). Zhu et al. (13, 14) proposed that the conformational changein IP3R induced by IP3 binding unmasks the putative sites thatfacilitate ubiquitin conjugation, resulting in degradation ofIP3R by the ubiquitin/proteasome pathway (71, 72). Alterna-tively, dissociation of IRBIT induced by IP3 binding may revealthe putative degradation signals or protease attack sites ofIP3R. Third, IRBIT may play the role of a linker moleculecoupling IP3R and other proteins to allow efficient signal prop-agation. Proteins possibly linked with IP3R include proteinswhose activities are regulated by Ca2� released from IP3R, orplasma membrane receptors, analogous with mGluR (36) andB2R (37). Indeed, substantial amounts of IRBIT were presentin the stripped microsome fraction (Fig. 2C), which might rep-resent IRBIT tightly bound to membrane proteins other thanIP3R. IP3 may disrupt these complexes, resulting in desensiti-zation of signals and/or translocation of linked proteins. Toidentify molecules possibly coupled with IP3R, we are nowsearching for IRBIT-interacting proteins. Fourth, IRBIT maybe a direct downstream signal transducer of IP3R1. It has beenthought that the only direct downstream molecule of IP3R1 isthe calcium ion, which acts on a wide variety of target mole-cules. Besides a multifunctional and universal second messen-ger like Ca2�, IP3R1 may utilize IRBIT as a downstream sig-naling molecule with more restricted target molecules thanCa2�. In this model, IRBIT released from IP3-bound IP3R1must be different (for example, in terms of phosphorylation2 H. Ando, unpublished observations.

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state) from IRBIT originally present in the cytosol, becausesignificant amounts of IRBIT already exist in the cytosol in thebasal state. In this respect, the model in which only phospho-rylated IRBIT binds to IP3R1 appears to be reasonable. Screen-ing of IRBIT-binding proteins may reveal the target moleculesof IRBIT.

Finally, the dissociation of IRBIT from IP3R in the presenceof IP3 is a feature which may be utilized for the development ofa new IP3 indicator based on fluorescence resonance energytransfer (FRET). FRET occurs when two fluorophores are inproximity and in the right orientation such that an exciteddonor fluorophore can transfer its energy to a second, acceptorfluorophore (73). Based on the cAMP-dependent dissociation ofcatalytic and regulatory subunits of cAMP-dependent proteinkinase, Adams et al. (74) developed a fluorescent indicator forcAMP. Similarly, Miyawaki et al. (75) reported a geneticallyencoded Ca2� indicator based on the Ca2�-dependent interac-tion between calmodulin and calmodulin-binding peptide. Al-though IP3 concentration changes could be detected by moni-toring translocation of the GFP-tagged pleckstrin homologydomain (76), a FRET-based IP3 indicator has yet to be devel-oped due to lack of suitable molecules. IP3-dependent dissoci-ation of IRBIT and IP3R1 is a characteristic that can provide anew tool allowing real-time imaging of the spatiotemporal dy-namics of IP3 concentrations in living cells, although furtherstudies focusing on the regulation of this interaction by phos-phorylation are needed.

In summary, we identified IRBIT, a novel IP3R1-interactingprotein, which was released from IP3R1 in the presence of IP3.Further studies aimed at elucidating the function of IRBIT,including the screening of IRBIT-interacting proteins, are an-ticipated to provide important insights into IP3/Ca2� signaling.

Acknowledgments—We thank M. Iwai for Sf9 expression and DNAsequencing, Dr. T. Uchiyama for providing GST-IbIIa, K508A, andR441Q proteins, Y. Makino at Okazaki National Research Institutes forprotein sequencing, and Drs. T. Michikawa and M. Hattori for criticalcomments on this manuscript.

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Hideaki Ando, Akihiro Mizutani, Toru Matsu-ura and Katsuhiko Mikoshiba Binding to the Receptor 3 Receptor upon IP3Released from the IP) Receptor-binding Protein, Is3IRBIT, a Novel Inositol 1,4,5-Trisphosphate (IP

doi: 10.1074/jbc.M210119200 originally published online January 13, 20032003, 278:10602-10612.J. Biol. Chem. 

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